Association of HLA Typing and Alloimmunity With Posttransplantation Membranous Nephropathy: A Multicenter Case Series.

RATIONALE & OBJECTIVES: Posttransplantation membranous nephropathy (MN) represents a rare complication of kidney transplantation that can be classified as recurrent or de novo. The clinical, pathologic, and immunogenetic characteristics of posttransplantation MN and the differences between de novo and recurrent MN are not well understood. STUDY DESIGN: Multicenter case series. SETTING & PARTICIPANTS: We included 77 patients from 5 North American and European medical centers with post-kidney transplantation MN (27 de novo and 50 recurrent). Patients with MN in the native kidney who received kidney allografts but did not develop recurrent MN were used as nonrecurrent controls (n = 43). To improve understanding of posttransplantation MN, we compared de novo MN with recurrent MN and then contrasted recurrent MN with nonrecurrent controls. FINDINGS: Compared with recurrent MN, de novo MN was less likely to be classified as primary MN (OR, 0.04; P < 0.001) and had more concurrent antibody-mediated rejection (OR, 12.0; P < 0.001) and inferior allograft survival (HR for allograft failure, 3.2; P = 0.007). HLA-DQ2 and HLA-DR17 antigens were more common in recipients with recurrent MN compared with those with de novo MN; however, the frequency of these recipient antigens in recurrent MN was similar to that in nonrecurrent MN controls. Among the 93 kidney transplant recipients with native kidney failure attributed to MN, older recipient age (HR per each year older, 1.03; P = 0.02), recipient HLA-A3 antigen (HR, 2.5; P = 0.003), steroid-free immunosuppressive regimens (HR, 2.84; P < 0.001), and living related allograft (HR, 1.94; P = 0.03) were predictors of MN recurrence. LIMITATIONS: Retrospective case series, limited sample size due to rarity of the disease, nonstandardized nature of data collection and biopsies. CONCLUSIONS: De novo and recurrent MN likely represent separate diseases. De novo MN is associated with humoral alloimmunity and guarded outcome. Potential predisposing factors for recurrent MN include recipients who are older, recipient HLA-A3 antigen, steroid-free immunosuppressive regimen, and living related donor kidney.

Uric acid increases cellular and humoral alloimmunity in primary human peripheral blood mononuclear cells.

AIM: Hyperuricaemia is common among kidney transplant recipients and has been associated with worse graft outcome. Since episodes of acute cellular rejection and chronic humoral rejection contribute to decreased graft survival, in this study the effect of uric acid on cellular and humoral alloimmunity was evaluated. METHODS: Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method in which humoral alloimmunity was induced in one-way MLR. Then the de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MRLs were used against resting PBMC similar to the stimulator cells of the above MLRs. RESULTS: Uric acid at a concentration above its crystallization threshold increased cellular proliferation in two-way MLRs. Supernatants from one-way MLRs performed in the presence of uric acid were more cytotoxic against PBMC from individuals that had conferred the stimulator cells for the above MLRs. CONCLUSIONS: Uric acid increases both cellular and humoral alloimmunity in human PBMC. These results offer a possible pathogenetic mechanism for the observed relation between hyperuricaemia and worse kidney allograft survival.

Indoleamine 2,3-dioxygenase suppresses humoral alloimmunity via pathways that different to those associated with its effects on T cells.

Chronic antibody-mediated rejection remains a major cause of late graft loss. Regarding cellular alloimmunity, the immunosuppressive properties of indoleamine 2,3-dioxygenase (IDO) have been well investigated; however, little is known of its effects on humoral alloimmunity. Therefore, the present study aimed to evaluate the effects of IDO on humoral alloimmunity. We developed a method for the induction of humoral alloimmunity in a one-way mixed lymphocyte reaction (MLR), which was measured with an antibody-mediated complement-dependent cytotoxicity assay using resting cells, which are similar to the stimulator cells of the aforementioned MLR. In parallel, cellular alloimmunity was assessed in two-way MLRs. The IDO inhibitor 1-methyl-DL-tryptophan was used for evaluating the role of IDO. In order to investigate whether the pathways known to serve a role in the effects of IDO on T cells are applied in humoral alloimmunity, the general control nonderepressible-2 (GCN-2) kinase activator tryptophanol and the aryl hydrocarbon receptor (AhR) inhibitor CH223191 were employed. The IDO inhibitor was revealed to increased cellular autoimmunity, but was decreased by the GCN-2 kinase activator. Unexpectedly, the AhR inhibitor decreased cellular alloimmunity. In addition, the IDO inhibitor was observed to suppress humoral alloimmunity, which may occur in manners independent of GCN-2 kinase AhR. The present study proposed that IDO may decrease humoral alloimmunity in primary human peripheral blood mononuclear cells via pathways that differ to those associated with its effect on T cells.

Assessment of Humoral Alloimmunity in Mixed Lymphocyte Reaction.

Humoral alloimmunity remains a significant and unresolved problem that constrains allograft survival. Thus, there is a need for the development of an easy, preferably non-radioactive, and inexpensive protocol for assessing the effect of various drug treatments on humoral alloimmunity. In order to satisfy this demand, we developed such a protocol in which de novo alloantibodies production is induced in one-way mixed lymphocyte reaction (MLR). The amount and capacity of the generated alloantibodies in the supernatant of each one-way MLR is assessed using an antibody-mediated cell dependent cytotoxicity (CDC) assay. The principle of the assay relies on the assessment of cellular survival of resting PBMCs isolated from the same donors, supplemented with the alloantibodies from the one-way MLR supernatant. The lesser is the cellular survival, the higher the production of alloantibodies in one-way MLR and consequently the more potent the humoral alloimmunity.

Characterization of the C1q-Binding Ability and the IgG1-4 Subclass Profile of Preformed Anti-HLA Antibodies by Solid-Phase Assays.

Humoral alloimmunity, particularly that triggered by preformed antibodies against human leukocyte antigens (HLA), is associated with an increased prevalence of rejection and reduced transplant survival. The high sensitivity of solid phase assays, based on microbeads coated with single antigens (SAB), consolidated them as the gold-standard method to characterize anti-HLA antibodies, ensuring a successful allograft allocation. Mean fluorescence intensity (MFI) provided by SAB is regularly used to stratify the immunological risk, assuming it as a reliable estimation of the antibody-level, but it is often limited by artifacts. Beyond MFI, other properties, such as the complement-binding ability or the IgG1-4 subclass profile have been examined to more accurately define the clinical relevance of antibodies and clarify their functional properties. However, there are still unresolved issues. Neat serum-samples from 20 highly-sensitized patients were analyzed by SAB-panIgG, SAB-IgG1-4 subclass and SAB-C1q assays. All 1:16 diluted serum-samples were additionally analyzed by SAB-panIgG and SAB-IgG1-4 subclass assays. A total of 1,285 anti-HLA antibodies were identified as positive, 473 (36.8%) of which were C1q-binding. As expected, serum-dilution enhanced the correlation between the C1q-binding ability and the antibody-strength, measured as the MFI (rneat = 0.248 vs. rdiluted = 0.817). SAB-subclass assay revealed at least one IgG1-4 subclass in 1,012 (78.8%) positive antibody-specificities. Among them, strong complement-binding subclasses, mainly IgG1, were particularly frequent (98.9%) and no differences were found between C1q- and non-C1q-binding antibodies regarding their presence (99.4 vs. 98.5%; p = 0.193). In contrast, weak or non-C1q-binding subclasses (IgG2/IgG4) were more commonly detected in C1q-binding antibodies (78.9 vs. 38.6%; p < 0.001). Interestingly, a strong association was found between the C1q-binding ability and the IgG1 strength (rIgG1dil = 0.796). Though lower, the correlation between the IgG2 strength and the C1q-binding ability was also strong (rIgG2dil = 0.758), being both subclasses closely related (rIgG1-IgG2 = 0.817). We did not find any correlation with the C1q-binding ability considering the remaining subclasses. In conclusion, we demonstrate that a particular profile of IgG subclasses (IgG1/IgG3) itself does not determine at all the ability to bind complement of anti-HLA antibodies assessed by SAB-C1q assay. It is the IgG subclass strength, mainly of IgG1, which usually appears in combination with IgG2, that best correlates with it.

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd-/-) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred "TCR75" CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2-/- recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd-/- recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2-/- recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a-/- TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd-/- recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity. This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

In human cell cultures, everolimus is inferior to tacrolimus in inhibiting cellular alloimmunity, but equally effective as regards humoral alloimmunity.

PURPOSE: Acute cellular rejection is the major cause of immune-mediated graft failure early in the course of kidney transplantation, whereas chronic antibody-mediated rejection is a major contributor to graft loss in the late post-transplant phase. Based mainly on the results of short-term studies, the calcineurin inhibitor tacrolimus prevails over the mammalian target of rapamycin (mTOR) inhibitors. However, the toxicity profile of the two drug categories differs, making the interchange between them appealing. In this study, the effect of tacrolimus and of the mTOR inhibitor everolimus on cellular and humoral alloimmunity was evaluated. METHODS: Cellular alloimmunity was assessed by cell proliferation in two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity, we developed a method in which humoral alloimmunity was induced in a one-way MLR. The de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MLRs were used against resting PBMC similar to the stimulator cells of the forementioned MLRs. Tacrolimus and everolimus were used at concentrations near their upper recommended trough levels. RESULTS: In two-way MLRs, tacrolimus inhibited cell proliferation more than everolimus. In one-way MLRs, tacrolimus and everolimus decreased alloantibody production to the same extent. CONCLUSIONS: In human cell cultures, everolimus is inferior to tacrolimus in inhibiting cellular alloimmunity, but equally effective as regards humoral alloimmunity. Thus, everolimus might be a safe alternative in case of tacrolimus toxicity, particularly after the early period of kidney transplantation.

A comparative analysis between proteasome and immunoproteasome inhibition in cellular and humoral alloimmunity.

Triggered by the successful administration of the proteasome inhibitor bortezomib in kidney transplant recipients with acute or chronic antibody-mediated rejection, we evaluated the effect of the proteasome inhibitor CEP-18770 and of the selective immunoproteasome inhibitor ONX-0914 on cellular and humoral alloimmunity. Cellular alloimmunity was assessed by cell proliferation in a two-way mixed lymphocyte reaction (MLR) with human peripheral blood mononuclear cells (PBMC). For assessing humoral alloimmunity we developed a method, where humoral alloimmunity was induced in one-way MLR. The de novo production of alloantibodies was measured with an antibody-mediated complement-dependent cytotoxicity assay, in which supernatants from the above MLRs were used against resting PBMC similar to the stimulator cells of the forementioned MLRs. In two-way MLRs ONX-0914 inhibited cell proliferation more than CEP-18770. In one-way MLRs CEP-18770 and ONX-0194 decreased alloantibody production to the same extent. Inhibition of the immunoproteasome is superior to inhibition of the proteasome in suppressing cellular alloimmunity, and equally effective as regards to humoral alloimmunity. Considering the selective expression of the immunoproteasome in immune cells and the expected restrictive toxicity of its inhibitors, these results render immunoproteasome an excellent target for the development of new immunosuppressive medications in the field of transplantation.